Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme's Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for the enzyme reaction. We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II␣ with phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme. This substitution reduced the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme. A similar but stronger effect was seen when the homologous mutation (Tyr 28 3 Phe) was introduced in yeast Top2. The ATPase activities of human TOP2␣(Tyr 50 3 Phe) and yeast Top2(Tyr 28 3 Phe) were resistant to both bisdioxopiperazines and the ATPase inhibitor sodium orthovanadate. Like bisdioxopiperazines, vanadate traps the enzyme in a salt-stable closed conformation termed the closed clamp, which can be detected in the presence of circular DNA substrates. Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP. Similarly, ADP trapped wild-type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes. Our results demonstrate that bisdioxopiperazine-resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme and that this difference in stability may lead to a loss of DNA-stimulated ATPase. We suggest that the DNA-stimulated ATPase of topoisomerase II is intimately connected with steps that occur while the N-terminal domain of the enzyme is dimerized.
The bisdioxopiperazines are catalytic inhibitors of eukaryotic type II DNA topoisomerases capable of trapping these enzymes as a salt-stable closed-clamp complex on circular DNA. The various bisdioxopiperazine analogs differ from each other because of structural differences in the linker connecting the two dioxopiperazine rings. Although the composition of this linker region has been found to be important for potency, the structural basis for this is largely unknown. To elucidate the role of the linker region in drug action, we have analyzed the effect of different linker substituents in otherwise identical analogs by studying their interaction with wild-type and mutant human topoisomerase II␣. Two mutations, L169I and R162Q, displayed differential sensitivity toward closely related analogs, suggesting that the linker region in these compounds plays a highly specific role in protein drug interaction. The finding that the L169I mutation, which probably represents a subtle structural change, was sufficient to confer resistance further emphases the importance of this region of the protein for bisdioxopiperazine inhibition of topoisomerase II. Comparing the sensitivity profiles of different bisdioxopiperazines against wildtype and mutant proteins with that of mitindomide, we observed a spectrum of sensitivity closely resembling that of ICRF-154, a bisdioxopiperazine with no linker substituents. We discuss the implications of these observations for the understanding of the mechanism of bisdioxopiperazine action on topoisomerase II.
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